Tissue plasminogen activator (TPA) is involved in the breakdown of non-specific blood clots in the blood circulation system. TNK is a derivative of TPA in which seven amino acids of the native sequence are modified so that the new molecule has changed specificity to the fibrin protein and changed pharmacokinetic properties leading to changed pharmacodynamic effects. TNK is a 527-amino acid glycoprotein of molecular weight of 70 KDa. It has been approved for use in the medicinal products for the treatment of acute myocardial infarction and some other thrombosis and embolism related breakage of blood vessels in different organs. Recently, it has been considered for the treatment of acute ischemic stroke and several clinical trials have been performed for this indication, but these trials have not given any conclusive evidence on the safe and effective dose of TNK for AIS.
Plasminogen activators are enzymes that activate the plasminogen to generate the serine protease plasmin that in turn degrades fibrin. Among the plasminogen activators used as drugs are: a) streptokinase [a bacterial protein], b) urokinase, [an enzyme synthesized in the kidney] and c) human tissue plasminogen activator [an enzyme produced by the vascular endothelium]. There are also other enzymes in development that are potential candidates as fibrinolytic drugs. The mechanisms of action of these activators differ: streptokinase forms a complex with plasminogen generating plasmin activity, urokinase cleaves plasminogen directly, and TPA forms a ternary complex with fibrin and plasminogen leading to plasminogen activation and clot dissolution in situ. Natural human TPA has a plasma half-life typically of 8-10 minutes. However, it was desired to have increased half-life so that the fibrinolytic therapy may be administrated effectively in a short period of time, with more beneficial efficacy outcomes. Therefore, several amino acid and deletion mutants of the protein were created and tested for the improved characters over TPA. TNK has six mutations that doubled half-life [to 20-24 min.] compared to tPA and improved characters such as higher fibrin clot specificity with concurrent less affinity for plasminogen activator inhibitor-1.
TNK was first disclosed in U.S. Pat. No. 5,385,732 wherein it was produced by recombinant DNA technology using an established mammalian cell line. Several other patents cover different aspects of the production and use of TNK, viz., U.S. Pat. No. 5,728,567, U.S. Pat. No. 5,714,145, U.S. Pat. No. 5,366,886, U.S. Pat. No. 5,094,953, U.S. Pat. No. 5,407,819 and U.S. Pat. No. 6,506,598. U.S. Pat. No. 5,407,819 discloses a method of the preparation of a TPA variant by displacement of a particular amino acid in the amino acid sequence. U.S. Pat. No. 5,612,029 discloses a variant of TPA, which is glycosylated at any positions on 103-105 and devoid of functional carbohydrate structure at position 117 of wild type human TPA sequence. U.S. Pat. No. 5,520,911 discloses the preparation of DNA sequences to encode the TPA variant. U.S. Pat. No. 5,424,198 discloses a method for the production of TPA by transforming the cells with mutant or wild-type DHFR genes in combination with tPA genes. These and several other documents also cover the various medical conditions or indications where TNK can be used as a drug for the treatment related to clot formation. One of the conditions covered by the US20080107641 is AIS. However, this application does not disclose any method of identifying a safe and effective dose of TNK for the treatment of AIS in human subjects. Instead it predicts the use of safe dose based on theoretical models of existing data matrices without giving any specific method of testing and qualifying the identified effective doses. Therefore, current prior art fails to address the unmet need for identification of safe and effective doses of TNK for the treatment of AIS and effective pharmaceutical compositions (formulations) for the treatment of AIS. The present invention addresses the above mentioned need and provides improved and more effective formulations of TNK for the treatment of AIS in human subjects, which is safe as well as easy to administer in a short period of time.
There are some studies that mention the use of TNK for the treatment of AIS, however, they fail to disclose any formulation of TNK with the amounts of TNK that are safe and effective as disclosed herein vis-à-vis its side effects. Besides, the TNK used for the preparation of formulations is prepared using the new technology of perfusion-based continuous fermentation system has not been used previously to prepare the TNK protein.
Various formulations are known that use TNK for the treatment of the AIS. However, so far none has been approved by any drug regulator that is safe and efficacious when vetted on risk-benefit analysis. Having identified this gap, some formulations of TNK are disclosed herein that have desired properties required of a safe and effective drug for the treatment of AIS. Some aspects of the method used in the present disclosure for the preparation of the TNK of high purity are known in the prior art. However, several new elements are used here that are not known in the art for the production, preparation, selection, testing and especially clinical qualification of the formulations of TNK for the treatment of AIS.